The present invention is in the field of phospholipase proteins that are related to the phospholipase D subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Phospholipases
There are three major families of known human phospholipase enzymes: Phospholipase A2, Phospholipase C, and Phospholipase D.
Enzymes in the Phospholipase A2 family (xe2x80x9cPlA2xe2x80x9d) hydrolyze the sn-2 fatty acid acyl ester bond of phosphoglycerides, releasing free fatty acids and lysophospholipids. The PlA2s constitute a diverse family of enzymes with respect to sequence, function, localization and divalent cation requirements. They play an important role in a variety of cellular processes, including the digestion and metabolism of phospholipids as well as the production of precursors for inflammatory reactions. The PlA2s have been classified into at least 5 groups (although different classification schemes exist and up to 10 groups have been identified by some authorities) based on their size, structure and need for divalent cations. Groups I, II and III all contain secreted forms of PlA, which are extracellular enzymes that have a low molecular mass and require calcium ions for catalysis. Groups IV and V contain cytosolic forms of PlA2s that have a high molecular mass and do not necessarily require calcium ions.
Amongst the best characterized of the PlA2 phospholipases are digestive enzymes secreted as zymogens by the pancreas. These enzymes, which are involved in the hydrolysis of dietary phospholipids, have strong homology to the venom phospholipases of snakes. Other PlA2s play important roles in the control of signaling cascades such as the cytosolic PlA2, Group IVA enzyme (xe2x80x9cPLA2G4Axe2x80x9d) which catalyzes the release of arachidonic acid from membrane phospholipids. Arachidonic acid serves as a precursor for a wide spectrum of biological effectors, collectively known as eicosanoids (and including the prostaglandin group of molecules) that are involved in hemodynamic regulation, inflammatory responses and other cellular processes.
Another biologically active phospholipid, platelet-activating factor (xe2x80x9cPAFxe2x80x9d) is hydrolyzed to metabolically-inactive degradation products by the group VII PlA2 known as PAF acetylhydrolase. Deficiency of PAF acetylhydrolase has been reported in patients with systemic lupus erythematosis and increased levels of PAF have been reported in children with acute asthmatic attacks. Elevated levels of the group II PlA2 known as PLA2G2A have been reported in plasma and synovial fluid in patients with inflammatory arthritis. Studies of a mouse colon cancer model showed that alleles of the murine ortholog of this gene were able to modify the number of tumors that developed in animals with multiple intestinal neoplasia (a mouse model of the human disorder known as familial adenomtous polyposis). Subsequent studies in humans showed mutations in PLA2G2A were associated with the risk of developing colorectal cancer. PLA2G2A is presumed to act through altering cellular microenvironments within the intestinal crypts of the colonic mucosa, although the precise mechanism by which this effect is exerted is not clear.
Enzymes in the Phospholipase C (xe2x80x9cPLCxe2x80x9d) family catalyze the hydrolysis of the plasma membrane phospholipids, phosphatidyl inositol phosphate (xe2x80x9cPIPxe2x80x9d) or phosphatidylinositol 4,5-biphosphate (xe2x80x9cPIP2xe2x80x9d), generating as products the second messengers, 1,4,5-inositol triphosphate (xe2x80x9cIP3xe2x80x9d) and 1,2-diacylglycerol (xe2x80x9cDAGxe2x80x9d). Molecules belonging to the PLC gene family are divided into subfamilies, PLC-beta, PLC-gamma and PLC-delta. PLC-delta is distinguished from PLC-gamma by lack of the SH2 and SH3 domains that are essential for activation of PLC-gamma by tyrosine protein kinases. PLC-delta is distinguished from PLC-beta by lack of the C-terminal region of PLC-beta that is responsible for binding and activation of G proteins. Various PLC enzymes play important roles in signal transduction cascades throughout the body. Activating signals include hormones, growth factors and neurotransmitters. One of the functions of IP2 is to modulate intracellular calcium levels while DAG is involved in the activation of certain protein kinases and can promote membrane fusion in processes involving vesicular trafficking.
Enzymes in the Phospholipase D (xe2x80x9cPLDxe2x80x9d) family catalyze the hydrolysis of phosphatidylcholine (xe2x80x9cPCxe2x80x9d) and other phospholipids to produce phosphatidic acid. A range of agonists acting through G protein-coupled receptors and receptor tyrosine kinases stimulate this hydrolysis. Phosphatidic acid appears to be important as a second messenger capable of activating a diverse range of signaling pathways. PC-specific PLD activity has been implicated in numerous cellular pathways, including signal transduction, membrane trafficking, the regulation of mitosis, regulated secretion, cytoskeletal reorganization, transcriptional regulation and cell-cycle control. Many proteins are attached to the plasma membrane via a glysylphosphatidylinositol (xe2x80x9cGPIxe2x80x9d) anchor. Phosphatidylinositol-glycan (xe2x80x9cPIGxe2x80x9d)-specific PLDs selectively hydrolyze the inositol phosphate linkage, allowing release of the protein.
Phospholipase D
The protein provided by the present invention is a novel human phospholipase splice form that is related to the phospholipase D (PLD) family. In particular, the novel phospholipase splice form provided by the present invention lacks exon 2 found in a prior art phospholipase protein (patent seq W57899). PLD proteins are known to exist as alternative splice forms. For example, alternate splice variants of two PLD isoforms, termed PLD1 and PLD2, have previously been identified (Steed et al., FASEB J. 1998 October; 12(13):1309-17).
The phospholipase D family is characterized by a conserved HXKXXXXD motif and this characteristic motif is essential for the catalytic function of PLD. A subclass of PLD exists that is characterized by a second HXKXXXXD motif with a conserved Asp to Glu substitution. PLD enzymes play important roles in signal transduction and membrane vesicular trafficking in mammalian cells (Pedersen et al., J Biol Chem Nov. 20, 1998; 273(47):31494-504). In particular, PLD cleaves phosphatidylcholine in response to cell stimuli, thereby releasing phosphatidic acid, which is involved in numerous cellular responses that may play a role in, for example, regulation of secretion, mitogenesis, or cytoskeletal changes (Steed et al., FASEB J. 1998 October; 12(13):1309-17).
The activity and regulation of recombinant human PLD2 are identical to that of recombinant mouse PLD2. Analysis of the amino acid sequences of the human PLD1 and PLD2 isoforms revealed Pleckstrin homology domains. (Steed et al., FASEB J. 1998 October; 12(13):1309-17). Orthologs of PLD may exist in vaccinia virus (Pedersen et al., J Biol Chem Nov. 20, 1998; 273(47):31494-504).
A murine PLD gene, termed sam-9 gene, has been found to be expressed at high levels in the brain, particularly in mature neurons of the forebrain, and the gene is turned on during late stages of neurogenesis (Pedersen et al., J Biol Chem Nov. 20, 1998; 273(47):31494-504).
Phospholipase proteins, particularly members of the phospholipase D subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of phospholipase proteins. The present invention advances the state of the art by providing previously unidentified human phospholipase proteins that have homology to members of the phospholipase D subfamily.
The present invention is based in part on the identification of amino acid sequences of human phospholipase peptides and proteins that are related to the phospholipase D subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate phospholipase activity in cells and tissues that express the phospholipase. Experimental data as provided in FIG. 1 indicates expression in humans in the brain (including neuroblastomas and infant brain), uterus, lung, ovary adenocarcinomas, and leukocytes.